Background
Pancreatic cancer (PC) is one of the most aggressive and lethal cancers, with an estimated 55,440 new cases and 44,330 deaths in United States in 2018 [
1]. The strong ability of local invasion and rapid metastasize are major hallmarks of PC, which contribute to the poor prognosis of patients. Thus, it is urgent to reveal the molecular mechanisms and target therapies toward the malignant biology and aggressive progression in PC.
Calreticulin (CRT), as a highly conserved endoplasmic reticulum (ER) Ca
2+-buffering chaperone, involves in various cellular processes [
2]. We previously reported that CRT overexpression promoted cell invasion, migration and drug resistance of PC by activating ERK/MAPK pathway [
3]. Most recently, we showed that CRT silencing inhibited EGF-induced epithelial-mesenchymal transition (EMT) via the Integrin/EGFR-ERK/MAPK pathway in PC [
4]. Based on previous studies, we next investigated the novel signaling pathways and molecular mechanisms involving the oncogenic role of CRT in PC development.
Endoplasmic reticulum stress (ERS) is a defensive response induced by various pathophysiological factors, which is triggered by three transmembrane signal transducers from unfolded protein response (UPR) family: PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α) and activating transcription factor-6 (ATF-6) [
5]. ERS plays a significant role in tumor biology including EMT mediated tumor invasion and metastasis [
6]. However, the definite role of ERS in malignancies remains controversial [
7]. Emerging evidences suggest that ERS plays a dual role in tumor progression. A transient ERS response activates a protective function and pro-survival pathway to cancers, whereas long term ERS triggers death signaling [
8,
9].
Intracellular free Ca
2+ is a multifunctional second messenger that controls diverse cellular functions [
10]. Recently, we have reported that alteration of CRT mediates intracellular free Ca
2+ concentration in PC cells [
4]. Moreover, dysfunction of cellular Ca
2+ homeostasis is a main stimulator of ERS [
11], which is closely related with cell invasion, immune evasion, EMT and drug resistance in various cancers [
12]. Therefore, we sought to evaluate the potential role of CRT in Ca
2+ homeostasis mediated ERS and EMT in PC, which, to our knowledge, has not been reported yet.
Materials and methods
Tissue samples and cell lines
This study was approved by the academic committee at the First Hospital of China Medical University. Written informed consent has been obtained from each patient. Eight-one pancreatic ductal adenocarcinoma (PDAC) tissues were procured from surgical resection specimens collected by the Department of Gastrointestinal Surgery at the First Hospital, China Medical University.
Human Capan-2 PC cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). SW1990 human PC cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured with recommended growth media with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA).
Fluo-3 assay
Thapsigargin (TG, Sigma, St Louis, MO, USA) is one of the key stimulators that cause acute ERS via specific inhibiting sarcoplasmic/endoplasmic reticulum Ca
2+-ATPases (SERCAs), resulting in an increase of cytoplasmic Ca
2+ concentration [
13]. The intracellular free Ca
2+ concentration was measured using Fluo-3 AM (Beyotime, Shanghai, China), according to the manufacturer’s instructions. Briefly, transfected PC cells were pretreated with 200 nM TG and 1% DMSO (control) for 4 h. Cells without or with TG treatment were subsequently loaded with 2 μM Fluo-3 AM for 30 min at 37 °C and then washed with Hanks’ Balanced Salt Solution (HBSS, Beyotime) for 3 times. Kept incubating with HBSS for 20 min, the fluorescence was visualized on a confocal microscopy (Leica Tcs Sp5 II, Leica, Heidelberg, Germany) at an excitation wavelength of 488 nm with an emission wavelength of 525 nm.
In addition, cells without or with TG stimuli were harvested by pancreatic enzymes without EDTA, washed by HBSS for 3 times, and then submitted to analysis by flow cytometry. Image analysis was performed using the Image J software. Each experiment was repeated 3 times.
Immunohistochemistry (IHC) assay
As described previously [
4,
14], 4-μm sections were covered with 0.3% H
2O
2, subjected to high pressure, added with goat serum, and then incubated with primary antibodies: CRT (Abcam, Cambridge, UK) and IRE1α (Cell Signaling Technology, CST, Beverly, MA, USA). Then the slices were incubated with the secondary antibodies, treated with streptavidin–peroxidase reagent, visualized with DAB, counterstained with hematoxylin and finally evaluated under microscope. The location of CRT and IRE1α in cytoplasm were considered for scoring. Staining intensity was scored as 0–3 (negative, weak, medium and strong). Extent of staining was scored as 0 (< 5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (> 75%) according to the positive staining areas to the whole carcinoma. The final scores were calculated by 3 pathologists. We used the same scoring method to evaluate the IHC assay in vivo and in human PDAC specimens.
Immunofluorescence (IF) staining
Capan-2 and SW1990 cell lines were implanted into 24-well culture plates covered with slices, fixed in 4% paraformal dehyde, permeabilized with Triton X-100 (0.5%) and incubated with 5% BSA. Then plates were incubated with the primary antibodies overnight: CRT (Abcam) and IRE1α (Cell signaling technology). The secondary antibodies (Proteintech, Chicago, IL, USA) were conjugated with FITC for CRT and TRITC for IRE1α. Hoechest33258 were used for nuclear visualizing.
Western blot (WB) assay
Whole protein lysates were prepared from transfected PC cells. Samples were loaded onto 10% SDS-polyacrylamide gels, transferred to PVDF membranes and incubated with primary antibodies: CRT (Abcam), IRE1α (CST), PREK (CST), phosphorylation PKR-like endoplasmic reticulum kinase (p-PERK, CST), ATF-6 (CST), ZO-1 (Proteintech), ZEB1 (Proteintech), N-cadherin (Proteintech), E-cadherin (Proteintech), Vimentin (Proteintech), phosphorylation extracellular regulated protein kinases (pERK, CST), extracellular regulated protein kinases (ERK, CST), X-box-binding protein 1 (XBP1, Proteintech), Snai1 (Proteintech), Slug (CST), Cavelino-1 (Proteintech), GAPDH (Proteintech) and β-actin (Proteintech) antibodies overnight at 4 °C. Then, membranes were incubated with secondary antibodies (Santa Cruz, CA, UK) and finally detected with an ECL detection kit (Thermo Scientific, Rockford, IL, USA). The experiments were repeated for 3 times.
Coimmunoprecipitation (CoIP) assay
CoIP was performed as before [
4,
14]. Briefly, PC cells were lysed in lysis buffer and the soluble supernatants were isolated. Magnetic beads (Bio-Rad, California, USA) were preincubated with primary CRT (Abcam), IRE1α (CST) or IgG (Santa Cruz) antibodies at 4 °C for 4 h with rotation. Then antibody-beads complexes were incubated with soluble supernatants at 4 °C overnight. Immunoprecipitated proteins were analyzed by WB with a variety of antibody.
Lentiviruses were synthesized by Genechem (Shanghai, China). PC cells were transfected with lenti-cas9 or lenti-sgRNA as described previously [
4,
14], and then screened using puromycin (Sigma). The stable sub-lines were subsequently transfected with sg1-CRT or sg2-CRT to specifically silence the target gene or an sgRNA control (scramble).
IRE1α siRNA and siRNA control were synthesized from GenePharma (Shanghai, China). Cells were transiently transfected with siRNA (20 μM) using oligofectamine3000 (Invitrogen, Carlsbad, CA, USA) as described by the protocol. All target sequences mentioned above were shown in Supplemental Material Table
1.
TG induced EMT construction
Stable transfected PC cells were treated with 200 nM TG or 1% DMSO (as a control) for 4 h. The EMT construction was verified by EMT-enhanced cell invasion and migration and EMT-induced changes in key proteins involving in EMT signaling.
Invasion and migration assays
Briefly, transfected PC cells (pretreated with TG or co-transfected with IRE1α) were plated in inserts that coated with matrigel (BD Biosciences, Sparks, MD, USA) in 24 well plates with FBS-free growth media. Growth media with 10% FBS was added to the bottom wells to generate a serum gradient. After 24 h, cells that had migrated to the underside of the inserts were stained with Crystal Violet Hydrate (Sigma). The migratory cells were counted in five random fields per well. The migration assay was done in a similar fashion without matrigel. Each experiment was repeated 3 times.
In vivo xenograft model
All animal work was performed in accordance with protocols approved by the Animal Care Committee of China Medical University. Total 15 nude mice (BALB/c-nu) were used. Transfected Capan-2 cells (1 × 10 [
6]) were respectively injected into bilateral axillae of 5 nude mice to construct subcutaneous tumor formation. Tumor volumes were calculated by the following formula: length × width × height × 0.52 in cm. Besides, transfected SW1990 cells (1 × 10 [
6]) were injected into the spleen of 10 nude mice to construct distant liver metastasis model, which were assessed by the number of liver metastases. These nude mice were killed 30 days later, and samples were extracted and fixed for hematoxylin and eosin (HE), and IHC staining.
Statistical analysis
Statistical analysis was performed using SPSS software 21.0 (Chicago, IL, USA). Continuous variables were expressed as the mean ± SD. The differences in intracellular free Ca2+ concentration, WB assay, cell migration and invasion assays and the number of liver metastases were compared through Student’s t-test. The differences of orthotopic tumor volumes were compared with paired sample t-test. Non-parametric and spearman correlation tests were analyzed for IHC assays in vivo and human PC samples. The association of target proteins expression with clinicopathological data was analyzed by Chi-squared. The Kaplan-Meier curve was used to estimate survival, and differences were analyzed by the log-rank test. P < 0.05 or P < 0.01 was considered significant.
Discussion
Due to the strong peripancreatic invasion and distant metastasis as well as insensitivity to chemotherapy, the prognosis of PC patients is extremely poor, with a 5-year survival rate of less than 5% [
25]. It is now well recognized that EMT is the “booster” for the malignant progression of PC [
26], which is implicated in enhancing invasion and metastasis in malignancies. EMT is typically characterized by the activation of ZEB1, N-cadherin, Vimentin, Snai1, Slug and Caveolin-1, and the downregulation of epithelial markers E-cadherin and ZO-1 expression [
27,
28]. Our previous study confirmed that CRT silencing inhibited EGF-induced EMT in PC via Integrin/EGFR-ERK/MAPK signaling [
4]. In present study, we first demonstrated that CRT mediated EMT via regulating intracellular free Ca
2+ mediated acute and chronic ERS in PC, which, to our knowledge, has not been reported yet.
CRT, initially identified as a ubiquitous ER protein in 1974 [
29], has diverse biological functions in cellular metabolism and biology, depending on the different locations inside and outside the ER [
30]. CRT regulates Ca
2+ homeostasis and molecular chaperoning activity within the ER [
31]. However, CRT located in the cytoplasm plays contradictory roles in cancer progression [
32]. For example, CRT exhibits an oncogenic role in lung [
33], breast [
34,
35], gastric [
36], hepatic [
37] and bladder cancers [
38], as well as in oral [
39] and esophageal squamous cell carcinoma [
40,
41], but acts as a tumor suppressor in neuroblastoma [
42,
43]. Meanwhile, the role of CRT remains inconclusive in colon [
44‐
47], prostate [
48,
49] and ovarian cancers [
50,
51].
Ca
2+ is mainly stored in ER lumen, which is a critical regulator involved in cancer progression [
52]. Accumulating evidences indicate that transient elevation of intracellular free Ca
2+ can promote tumor cell migration and invasion. Conversely, sustained free Ca
2+ stimulation might lead to the cell apoptosis and death [
53]. The disruption of Ca
2+ homeostasis also triggers ERS, that is closely associated with EMT [
54]. For example, EMT is induced in breast cancer cells in parallel with the increase of cytosolic Ca
2+, whereas chelating Ca
2+ blocked the induction of EMT markers [
55]. CRT is considered as an intracellular Ca
2+ regulator. It contains two Ca
2+-binding domains: C-domain with a low affinity and high capacity region, and the P-domain with a high affinity and low capacity region [
56]. Thus, CRT deficiency generally leads to the decrease of intracellular Ca
2+ storage [
57,
58]. However, to our knowledge, there is no direct research involving the mechanism of CRT in regulating Ca
2+-mediated EMT in PC. TG, as an effective inhibitor of SERCAs, causes an increase of cytoplasmic free Ca
2+ concentration and further induces acute ERS via the depletion of Ca
2+ from ER [
59]. We first found that CRT silencing inhibited TG-induced increase of intracellular free Ca
2+ concentration. Meanwhile, TG-induced EMT in vitro by activating the key protein targets in EMT and ERK/MPK signaling (Slug, E-cad, ZO-1 and pERK), and enhancing cell mobility, which was also reversed by CRT silencing. Slug (also known as Snail2), is the most thoroughly investigated EMT regulator [
60]. As a transcription factor, Slug binds to the E-cadherin promoter to repress its transcription and triggers the steps of desmosomal disruption and cell spreading, which is the key step of the EMT process [
61]. ERK signaling is also essential for EMT. An ERK-dependent epigenetic remodeling of regulatory elements results in a gene expression programme essential for driving EMT [
62]. TGF-β1 activates ERK signaling, which is required for TGF-β1-mediated EMT in vitro [
63]. Musashi2 promotes EGF-induced EMT in PC via ZEB1-ERK/MAPK signaling [
64]. Taken together, CRT silencing inhibited TG-induced acute ERS and EMT via regulating Slug and ERK signaling in vitro. Interestingly, TG also induced IRE1α expression which was negatively regulated by CRT in vitro. IRE1α silencing enhanced TG-induced cell mobility. Thus, we next focused on the relationship between CRT and UPR in chronic ERS.
Chronic ERS produces endogenous or exogenous damage to cells and triggers an UPR response. IRE1α is the most evolutionally conserved one in UPR [
65]. As an ER type I transmembrane protein, the role of IRE1α in cancers is no longer simply considered as an oncogene or tumor suppressor, but a key component of cell fate switch, depending on different cancer types [
66]. IRE1α mediated apoptosis in human non-small cell lung cancer (NSCLC) A549 cells induced by a Tetramethylpyrazine analogue [
67]. However, IRE1α overexpression was associated with the resistant mechanism to osimertinib in NSCLC HCC827/OSIR Cells [
68]. Similarly, several studies have shown that IRE1α plays a contradictory role in colon cancer cells [
69‐
72]. We next found that CRT was co-immunoprecipitated and co-localized with IRE1α in vitro and its stable silencing caused chronic ERS by specifically activating IRE1α independent of IRE1/XBP1 axis. It is well known that IRE1/ XBP1 axis plays a key role in mediating UPR in response to ERS [
73]. IRE1α/XBP1 pathway is a potential therapeutic target for Myc-driven cancers and multiple myeloma [
74,
75]. However, IRE1 also exhibits XBP1-independent biochemical activities just shown in current study and previous reports [
76,
77]. We next found that IRE1α silencing promoted EMT in vitro by enhancing cell mobility and activating EMT and ERK signaling, which was significantly reversed by CRT silencing. Interestingly, ERK1/2 activation is partially IRE1-dependent in mouse embryonic fibroblast cells treated with ER stress inducer [
78], while IRE1 silencing attenuated ERK1/2 activation following ER stress in gastric cancer cells [
79], which is inconsistent with current study. These inconsistent results might be due to the different cell types and microenvironment. Taken together, CRT silencing inhibited IRE1α silencing-induced chronic ERS and EMT via Slug and ERK signaling in PC cells, which has not been reported, to our knowledge.
Finally, CRT silencing inhibited subcutaneous tumor size and distant liver metastasis in vivo following with the increase of IRE1α expression. In human PC samples, CRT overexpression and IRE1α positive expression was positively and negatively associated with advanced clinical progression and poor survival of PC patients, respectively. Additionally, we found a negative expression of CRT and IRE1α in PC samples, which coordinately affected the patients’ survival. These findings indicate that CRT and ERS pathways cooperatively contribute to the aggressive progression of PC.
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